U.S. patent number 4,594,244 [Application Number 06/579,117] was granted by the patent office on 1986-06-10 for antigenic materials.
This patent grant is currently assigned to Council of Governors of the United Medical and Dental Schools of Guy's. Invention is credited to Abu S. M. Giasuddin, Thomas Lehner.
United States Patent |
4,594,244 |
Lehner , et al. |
June 10, 1986 |
Antigenic materials
Abstract
An antigenic material having a molecular weight of 3800-4500,
and useful in the preparation of an anticaries vaccine is produced
from a known antigenic material, antigen I/II of molecular weight
185,000 daltons obtained from the culture supernatant of
Streptococcus mutans. The smaller molecular weight fraction of the
invention contains the same antigenic determinants as the known
185,000 dalton material but the lower molecular weight material can
be used with less side effects. The antigenic material can be used
for the production of vaccine preparations that could be
administered parenterally or by topical application to the
gums.
Inventors: |
Lehner; Thomas (Barnet,
GB2), Giasuddin; Abu S. M. (Zaria, NG) |
Assignee: |
Council of Governors of the United
Medical and Dental Schools of Guy's (London,
GB2)
|
Family
ID: |
10537943 |
Appl.
No.: |
06/579,117 |
Filed: |
February 10, 1984 |
Foreign Application Priority Data
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Feb 14, 1983 [GB] |
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8303994 |
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Current U.S.
Class: |
424/150.1;
530/350; 530/825; 435/71.2; 530/806; 424/165.1; 424/244.1 |
Current CPC
Class: |
C07K
16/1275 (20130101); C07K 14/315 (20130101); Y10S
530/806 (20130101); Y10S 530/825 (20130101); A61K
39/00 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
14/195 (20060101); C07K 14/315 (20060101); C07K
16/12 (20060101); A61K 38/00 (20060101); A61K
39/00 (20060101); A61K 039/09 (); A61K 039/40 ();
C12P 021/00 () |
Field of
Search: |
;260/112R,112B
;424/88,177,87,92 ;435/68,885 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0009872 |
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Apr 1980 |
|
EP |
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0068660 |
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Jan 1983 |
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EP |
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0116472 |
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Aug 1984 |
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EP |
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2060647 |
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May 1981 |
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GB |
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Other References
J of Immunology, 135, 1-6 (1985), Lehner et al. .
Journal of General Microbiology (1979), 114, 109-151, Russell.
.
Infection and Immunity, vol. 29, No. 3, 1980, pp. 999-1006, Russell
et al. .
Infection and Immunity, vol. 28, No. 2, 1980, 489-493, Russell et
al. .
Biological Abstracts 73, Abstract 39207, 1982..
|
Primary Examiner: Schain; Howard E.
Attorney, Agent or Firm: Oblon, Fisher, Spivak, McClelland
& Maier
Claims
We claim:
1. Antigen X having the following characteristics:
1. It has a molecular weight as determined by SDS-PAGE in the range
of 3,800-4,500 daltons,
2. It is immunogenic in experimental animals, forming precipitating
antibodies,
3. It reacts with antisera raised against antigen I, antigen I/II
or antigen II as well as with antisera raised against itself, but
does not react with antisera raised against antigen III,
4. It is distinct from serotype polysaccharide antigens, glycerol
teichoic acid, dextran and similar glucans, and does not synthesise
glucans from sucrose,
5. It is substantially of a proteinaceous nature,
6. It absorbs ultra-violet light strongly between 220 and 250 nm
(maximum at 230 nm),
7. Its immunogenic activity is destroyed by the enzyme pronase.
8. It is substantially free from antigen I/II.
9. It induces production of helper T cells but not of suppressor T
cells.
2. Antigen X according to claim 1 substantially free from antigenic
materials having a molecular weight, determined by SDS-PAGE, less
than 3800 daltons or greater than 4500 daltons.
3. Antigen X according to claim 1 obtained from the culture
supernatant of Streptococcus mutans.
4. An anti-caries preparation comprising antigen X according to
claim 1 together with a solid or liquid carrier.
5. A preparation according to claim 4 in a form suitable for
parenteral or topical administration.
6. An antibody against antigen I/II that has been raised in a host
that has been injected with antigen X according to claim 1.
7. A monoclonal antibody according to claim 6.
8. An anti-caries preparation comprising an antibody according to
claim 6 together with a solid or liquid carrier.
9. A process for the production of antigen X as defined in claim 1
comprising subjecting antigen I/II obtained form culture
supernatant of Streptococcus mutans in an excess quantity to
polyacrylamide gel electrophoresis using a urea containing gel, and
isolating from the gel fraction having a molecular weight in the
range 3800-4500 daltons.
10. A process for the production of antigen X as defined in claim 1
comprising heating antigen I/II obtained from culture supernatant
of Streptococcus mutans, subjecting the heated material to gel
filtration and isolating from the gel a fraction having a molecular
weight in the range 3800-4500 daltons.
11. A process according to claim 8 or 9 wherein the 3800-4500
daltons molecular weight fraction is subjected to polyacrylamide
gel electrophoresis using a gel containing 15% w/w polyacrylamide
and also containing urea and isolating a fraction of molecular
weight 3800 daltons.
12. A process for the production of antigen X as defined in claim 1
comprising subjecting antigen I/II obtained from culture
supernatant of Streptococcus mutans to treatment with a bacterial
protease, subjecting the resulting material to polyacrylamide gel
electrophoresis using a urea containing gel and isolating a
fraction of molecular weight 3800-4500.
13. An anti-caries preparation comprising an antibody according to
claim 7 together with a solid or liquid carrier.
14. A method for the treatment of a human or animal host to protect
the host against dental caries which comprises administering to the
host a preparation according to any one of claims 4, 5, 8 or 13.
Description
DESCRIPTION
This invention relates to antigenic materials suitable for use in
vaccines against dental caries.
The antigenic components of Streptococcus mutans have been
extensively studied since it was recognised that this was the major
organism responsible for the development of dental caries. It has
previously been recognised that immunisation with whole cell or
cell-wall preparations of S. mutans may produce undesirable side
effects and hence there has been a desire to produce vaccines
containing one or more pure specific antigens to confer the
necessary protection against dental caries.
Four predominantly protein antigens (designated I to IV) have
previously been identified in the culture supernatant of S. mutans
by immunodiffusion and immunoelectrophoresis against corresponding
rabbit antisera (see Archs Oral Biol. 23 7-15, Russell and Lehner).
Antigen I referred to in that paper, which is now known as antigen
I/II, has a molecular weight, as determined by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in the range
of 175,000 to 195,000 daltons. Glycoproteins with similar molecular
weights to antigen I/II have been isolated by other workers, as has
a glycoprotein with a molecular weight of 29,000 daltons (see J.
Gen. Micro. 114 109-115, R. R. B. Russell). Further investigations
on the antigen I/II molecule (MW 185000 daltons) has shown that
antigen I (MW 150,000 daltons) and II (MW 48,000 daltons) are
associated in this single molecule (see UK Patent Publication No.
2060647A and Infection and Immunity 28 486-493, M. W. Russell et
al). This latter reference also indicates that antigen II can be
separated and isolated by pronase digestion of antigen I/II
followed by column chromatography, while it is disclosed that
antigen I can be isolated from antigen I/II by affinity
chromatography (see Infection and Immunity 29 999-1006, M. W.
Russell et al).
There has now been found, in accordance with the present invention,
an antigenic substance derivable from S. mutans having a molecular
weight as determined by SDS-PAGE in the range of 3,800-4,500
daltons. Surprisingly, despite the low molecular weight of this
material, it is found to contain both the antigenic determinants I
and II which are present in the material of 185,000 daltons.
The low molecular weight antigen, which will hereinafter be
referred to as antigen X, has certain characteristics as listed
below. The molecular weight in conjunction with some or all of the
other characteristics may be used in identifying the material of
the present invention:
1. It has a molecular weight as determined by SDS-PAGE in the range
of 3,800-4,500 daltons,
2. It is immunogenic in experimental animals, forming precipitating
antibodies,
3. It reacts with antisera raised against antigen I, antigen I/II
or antigen II as well as with antisera raised against itself, but
does not react with antisera raised against antigen III,
4. It is distinct from serotype polysaccharide antigens, glycerol
teichoic acid, dextran and similar glucans, and does not synthesise
glucans from sucrose,
5. It is substantially of a proteinaceous nature,
6. It absorbs ultra-violet light strongly between 220 and 250 nm
(maximum at 230 nm),
7. Its immunogenic activity is destroyed by the enzyme pronase.
The present invention relates not only to the above antigen X but
also antigenic substances which are of equivalent immunological
activity to antigen X. In the Example which follows, an amino acid
analysis is given for the 3,800 dalton peptide prepared as
described in the Example. It will be appreciated, however, that the
invention is not restricted to a material having precisely this
composition, nor precisely the amino acid sequence of the natural
product, where modifications in the amino acid composition and
sequence do not alter the immunogenic activity as discussed
below.
Antigen X is isolable from S. mutans though it need not necessarily
be obtained from this source, or indeed from a Streptococcus
organism. As illustrated in the Example which follows, antigen X
can be obtained from a starting material comprising the 185,000
dalton antigen termed antigen I/II. Antigen II of 48000 daltons can
be separated and isolated by pronase digestion of antigen I/II
followed by column chromatography, while antigen I of 150000
daltons can be isolated by affinity chromatography. Antigen X of
the present invention, while being of much smaller size, than
either antigen I or antigen II, still retains both antigenic
determinants I and II. Without wishing to be bound by any theory,
it is suggested that the 185,000 dalton material may contain
repeating units of I/II antigenic determinants of which the
smallest unit corresponds to antigen X of the present invention. If
that is the case, it is possible either that S. Mutans synthesises
antigens with these two determinants in a range of molecular sizes,
or that the 185,000 dalton material is broken down by the
streptococcal proteases inside the cells or in the extra cellular
fluid.
When antigen X is to be isolated from the organism, it can be
prepared using a starting material of antigen I/II prepared by any
previously known method. A particular method is indicated in the
following Example and reference is also made to the method in
British Patent publication 2060647A. In broad terms, antigen X can
be obtained from antigen I/II either by subjecting it in an excess
quantity to polyacrylamide gel electrophoresis using a urea
containing gel, isolating from the gel a fraction having a
molecular weight in the range of 3800-4500 daltons. Overloading of
the 185,000 dalton preparation e.g. at least a ten-fold excess of
the normal quantity, on 15% SDS-PAGE in the presence of 6M-urea
revealed the presence of the antigen of the present invention which
was not evident when normal quantities (20 .mu.g protein/cm slot)
were used. A convenient overload figure is 280.+-.30 .mu.g
protein/cm slot. Alternatively, antigen I/II can be heated and the
heated material subjected to gel filtration when antigen X can be
isolated.
Although the technique using SDS-PAGE as described in the Example
part d (i) enables a satisfactory preparation of antigen X, it has
also been found possible to produce a single band of 3,800 dalton
material by boiling the starting antigen I/II in 4% SDS buffer for
seven minutes to dissociate and release the components and then
sieving it through Sephacryl S-200 with the same buffer (see part d
(ii) in the Example which follows). The fact that antigen X can be
recovered after denaturing antigen I/II from which it is prepared
suggests that the three dimensional antigenic structure must have
been restored in the final product to that resembling the starting
material. However, as is illustrated in the Example, pronase
digested both the antigenic determinants of the 3,800 peptide
within thirty minutes, which differs from the result with the
185,000 dalton material in which antigen I was readily digested but
antigen II remained intact even after 16 hours of digestion. This
suggests that both antigenic determinants I and II may be exposed
to enzyme action in antigen X whereas in the larger molecule
antigen II may be concealed.
Antigen X has been found to have two amino-terminal residues,
glycine and alanine, which suggests either that the preparation is
an equimolar mixture of two peptides with very similar molecular
weights or that the peptide is made up to two chains linked by a
disulphide bridge. However, as the molecular weight of the peptide
is unchanged after treatment with mercaptoethanol, it is more
likely that the low molecular weight peptide is a mixture of two
separate chains, though the invention is not to be bound by this
theory.
The carbohydrate content of antigen X has been analysed in terms of
the total monosaccharide concentration and the results are given in
part e (iii) of the Example. The findings suggest that the
carbohydrate content of antigen X is negligible, as a 2% level
would allow for carbohydrate corresponding to a molecular weight of
only 80 daltons, and the two samples tested gave percentages of
1.64 and 2.08. Antigen X also differs from antigen I/II in failing
to show lipid content ad determined by thin layer chromatography.
It therefore seems that antigen X is a protein with no lipids and
only a trace of carbohydrate which need not be present especially
if the antigen is obtained from sources other than S. mutans.
The present invention provides antigen X either in substantially
pure form or associated with other materials. It may, for example,
be mixed with other antigens of different immunogenicity but is
preferably completely free from cells and cell-wall fragments. Most
preferably, it is in substantially pure form such that on SDS-PAGE
it exhibits a single protein band. If necessary, affinity
chromatography may be used to achieve the desired purity of the
product.
Antigen X from sources other than S. mutans may have an amino acid
sequence corresponding exactly to the product from that source or,
alternatively, may be formed by altering amino acids within the
sequence where these changes do not affect the immunogenic
activity. The alteration can take the form of an omission or
addition of one or more amino acids and/or a modification of one or
more amino acids. Such changes are permitted provided the product
has unaltered immunogenic activity. While such changes in the
antigen would, perhaps, be detectable by monoclonal antibodies
raised against the natural product, this is not considered to be an
indication that their immunogenic activity is altered. Equivalent
immunogenic activity is shown by the fact that antibodies produced
by the modified antigen X will neutralise the same bacterium as the
natural product and by the ability of the modified antigen X to
combine in vitro with antibodies produced by the natural
product.
Particular immunogenic equivalents may be formed by modifying
reactive groups within the natural sequence or, particularly, the
N-terminal amino groups and/or the C-terminal carboxyl groups.
Other equivalents include salts formed with acids and bases,
particularly physiologically acceptable inorganic and organic acids
and bases. Esters and amides may also be formed with the carboxyl
group. Such modifications of the antigen are preferably carried out
where they enable the production of a more stable active peptide
which is less susceptible to enzymic breakdown in vivo.
It is preferred to place the antigen in as similar a conformation
or conformational environment as possible to that which it occupies
in vivo. The precise structure of the antigen is not known, though,
as indicated above, it is believed to comprise two separate but
similar peptide chains. It may therefore be appropriate to
introduce crosslinking into the material in order to stabilise it
and this may possibly be achieved by suitable replacement of amino
acids by ones which are capable of covalently bonding with others
in the other chain; in particular, cysteine may be introduced to
form a disulphide bond with another chain. Alternatively, it may be
appropriate to loop the peptide by linking together ends of chains,
e.g. with an amide link between chain termini.
Antigen X of the present invention is useful for providing
protection against dental caries. This in vivo production of
antibodies specific to the antigenic determinants I and II on
antigen X is believed to be an important aspect of their action. It
is for this reason that the present invention also extends to
antigenic substances of equivalent immunological activity.
Vaccines containing antigen X or immunogenic equivalents may be
prepared by conventional methods and administered by various
routes. They will usually be in a form suitable for injection or
for oral administration. Thus, the antigen may for example be
formulated in a diluent or on a solid carrier. The injectible
solutions will usually be given subcutaneously or intramuscularly.
Oral methods of administration may produce a effect systemically or
locally in the mouth, and orally active preparations can be
formulated as a gel, toothpaste, mouthwash or chewing gum.
Although the vaccines will usually be given to produce protection
against attack of dental caries, it is also envisaged that the
vaccine may be given to a patient already having caries. As
previously indicated, antigen X may also be mixed with other
antigens of different immunogenicity; this may be necessary to
ensure that antibodies reacting with all serotypes of S. mutans are
produced by the vaccines.
The antibodies to antigen X and its immunogenic equivalents also
form part of the present invention. Thus, while the antigen of the
present invention may be given to a patient to induce the
production of antibodies, the antibodies themselves may be given
directly for use in passive immunisation where this seems
appropriate. Such antibodies may be prepared by the general method
for preparing antisera given in section (b) of the Example which
follows. Alternatively, monoclonal antibodies can be prepared by
the genera technique of Kohler and Milstein in which, for example,
a mouse host is injected with antigen X of the present invention,
spleen cells from the immunised host mouse isolated and hybridised
with myleoma cells and appropriate hybridomas isolated that will
produce monoclonal antibodies that will subsequently protect a host
against dental caries. Antibodies, including monoclonal antibodies,
can be formulated for passive immunisation as indicated above for
the formulation of antigen X including the solid or liquid
formulations such as gels, toothpastes, mouthwashes or chewing
gums.
Dosage levels are selected in order to give high levels of
protection and will generally be lower than those envisaged for
antigen I/II. As illustrated in section (f) of the Example which
follows, antigen X is capable of producing a significant response
with only 10 .mu.g of protein and using Allugel as adjuvant. One
reason why smaller amounts of material may be effective is that
antigen X apparently increases the production of only the helper
T-cells and not the suppressor T-cells. A suitable dosage for human
immunisation by the subcutaneous route may conveniently be of the
order of 0.01 to 0.5 mg given with aluminium hydroxide or another
suitable adjuvant. A frequency of administering the vaccine to
young patients will conveniently be: 6 months, 2 years, 5 years and
10 years, with the initial dose being accompanied by adjuvant and
the subsequent doses being administered without adjuvant and being
about 1/2 to 1/4 the level of antigen in the initial dose. The
frequency of administration can, however, be determined by
monitoring the antibody levels in the patient.
The present invention accordingly also provides a pharmaceutical
composition comprising antigen X, an immunogenic equivalent thereof
or an antibody to either of these, in combination with a
physiologically acceptable diluent or carrier. In addition, it
provides a method of treating a mammalian, particularly a human,
patient, in order to provide at least some protection against
dental caries by administering to the patient a composition as
defined above.
The antigen of the present invention may also be used in assaying
antibodies to antigenic determinants I and II, e.g. where these are
being monitored to determine the level and timing of vaccination
needed. For this purpose, the antigen will normally be attached to
an inert carrier such as a dextran, e.g. Sepharose. Alternatively,
the antigen may be used in affinity chromatography in order to
isolate such antibodies.
Like antigen I/II, antigen X of the present invention is capable of
eliciting the production of antibodies to antigens I and II giving
a high level and breadth of protection against dental caries.
Antibodies to antigen X can be completely absorbed by the antigen
I/II and are indistinguishable from those induced by antigen I/II
itself. It has the added advantages, however, that in being
smaller, it is better defined and hence less likely to give rise to
side effects, and also it activates T-helper cells but not
T-suppressor cells.
The following Examples illustrate the invention. Example 1
describes a typical method for preparing antigen X from naturally
occurring materials and gives details of its properties and
activity.
EXAMPLE 1
Preparation of Antigen X
(a) Culture of S. mutans
Streptococcus mutans serotype c (Guy's strain) was grown in 12
liters of pre-warmed Todd-Hewitt broth (Oxoid) at 37.degree. C.,
using an overnight culture in 100 ml of Todd-Hewitt broth as an
inoculum. The growth was continued for 60-65 hours. The culture
supernatant was separated by centrifugation in a continuous flow
rotor at 37,000 g. (Guy's strain of S. mutans serotype C is a
typical strain of serotype C of S. mutans and many similar strains
of serotype C of S. mutans are available from Culture Collections
including the ATCC in USA, e.g. ATCC 27607
(b) Extraction and purification of Antigen I/II
The protein antigens in the resulting culture supernatant were
precipitated with 75% ammonium sulphate. The precipitate was spun
down, dissolved in urea-tris buffer, dialysed against water and
chromatographed on a diethylaminoethyl (DEAE)--cellulose column
(Whatman DE52,30.times.1.5 cm). The column was eluted with 6M
urea--0.01M tris buffer (ph 8.0) containing 0.05M sodium chloride
and the fractions were tested in single radial immunodiffusion
(SRID) against antiserum to antigen I/II (prepared as described
below). The positive fractions were pooled, dialysed against water,
lyophilised and then dissolved in 1% ammonium bicarbonate and gel
filtered on a Sepharose 6B column (Pharmacia, Great Britain, Ltd,
90.times.2.5 cm); with the same buffer. The eluate was monitored at
280 nm and 3 ml fractions were tested by SRID against antiserum to
I/II. The fractions containing antigen I/II were pooled and
lyophilised.
Antisera for use in SRID described above were raised in New Zealand
white rabbits by intramuscular injections of 1 mg of antigen I/II
in Freund's complete adjuvant, followed three weeks later by
subcutaneous injection of 1 mg antigen I/II in Freund's incomplete
adjuvant. Blood was taken three or more weeks after the last
immunisation. Single radial immunodiffusion was used for the
identification of antigens in the fractions in 1.0% agarose gel
containing 1-2% antiserum in veronal buffer (pH 8). (Other antisera
used in this Example were also prepared by this method.)
(c) Detection of Antigens by SDS-PAGE
High molecular weight antigens were detected by SDS-PAGE on 7.5%
polyacrylamide gels in a vertical slab gel apparatus as described
previously (see UK Patent publication 2,060,647A). Low molecular
weight antigens were detected by 15% SDS-PAGE in the presence of
6M-urea (Bethesda Research Laboratories (BRL), USA, Biologue 1981).
The 15% resolving gel contained 0.1M-sodium phosphate buffer (pH
7.2), 0.1% SDS, 0.02% sodium azide and 6M-urea, and a 7.5%
polyacrylamide stacking gel in the same buffer was used. The gel
was prepared to provide sample wells as described in UK Patent
publication 2060647A and was overloaded with the antigen I/II
preparation prepared in step (b) above (280.+-.30 .mu.g protein/cm
slot). Gels were run overnight at 70 V and stained with Coomassie
Brilliant Blue.
A pre-stained protein molecular weight standard mixture (BRL, USA),
containing ovalbumin (43000), .alpha.-chymotrypsinogen (25700),
.beta.-lactoglobulin (18400), lysozyme (14300), cytochrome c
(12300), bovine trypsin inhibitor (6200) and insulin A and B chains
(3000), were used to determine the molecular weights of the
antigens. A number of components were detected having a lower
molecular weight than antigen I/II itself; in 27 out of 29
preparations of the starting antigen I/II a low molecular weight
peptide having a molecular weight which varied from 3800 to 4500
daltons was detected.
(d) Purification of low molecular weight antigens
(i) SDS-PAGE
A sample of 2.5 mg per gel of the starting antigen I/II prepared as
under (b) above was loaded, after equilibration with the sample
buffer, on 15% polyacrylamide--6M urea gels and electrophoresed as
described in section (c) above, using 3 different quantities of
antigen I/II. After electrophoresis, the gel was sliced into five
sections according to the molecular weight ranges (1)>43000,
(2)<43,000>25,700, (3)<25,700>18,400,
(4)<18,400>6,200 and (5)<6,200. The slices were minced
separately by forcing each through a hypodermic syringe and the
antigens were then extracted three times with 0.01M tris-HCl buffer
(pH 8.0), containing 0.05% SDS and 1 mM phenyl methylene sulphonyl
fluoride (PMSF) at 37.degree. C. for 36-48 hours. The three
extracts from each slice were pooled, passed through a glass fibre
filter under vacuum, dialysed extensively against water at
4.degree. C. and lyophilised. The dialysis tubing used had a
molecular weight cut-off of 1000 daltons. The lyophilised material
was reconstituted in 0.85 % NaCl, spun for 3 minutes at 25,000 g
and the supernatant was collected.
The proteins and peptides eluted from each of the 5 gel slices were
assayed for their protein content by the method of Lowry et al (see
J. Biol. Chem 193 265-275, (1951)) with bovine serum albumin as
standard. They were also assayed for their antigenicity with
antisera to antigen I/II, I, II and III by SRID, the antisera all
being produced as previously indicated for antisera to antigen
I/II. The results are given in the following Table 1 for the three
different loadings of antigen I/II.
TABLE 1
__________________________________________________________________________
Quantity of Antibodies to antigen I/II Fraction Molecular Protein
Content antigens loaded (mg) No. Weight .mu.g % Yield I II III
__________________________________________________________________________
5.0 1 >43000 1330 26.60 + + + 2 <43000 > 25700 190 3.80 +
+ + 3 <25700 > 18400 178 3.05 + + Trace 4 <18400 > 6200
126 2.52 + + - 5 <6200 198 3.96 + + - 5.7 1 >43000 1230 24.60
+ + + 2 <43000 > 25700 180 3.60 + + + 3 <25700 > 18400
165 3.30 + + Trace 4 <18400 > 6200 110 2.20 + + - 5 <6200
180 3.60 + + - 6.5 1 >43000 1211 24.22 + + + 2 <43000 >
25700 152 3.04 + + + 3 <25700 > 18400 158 3.16 + + Trace 4
<18400 > 6200 106 2.12 + + - 5 <6200 157 3.14 + + -
__________________________________________________________________________
+ presence of the antigenic determinant - absence of the antigenic
determinant
As can be seen from the above Table 1, as the amount of antigen
loaded was increased, so the percentage yield of various fractions
decreased. Antigenic determinant I/II was present in all fractions
in each case, including the lowest molecular weight fraction
(fraction 5) which comprised predominantly antigen X of M.W.
3800-4500. A variable amount of antigenic determinant III was also
detected in fractions 1, 2 and 3 but not in 4 or 5.
(ii) Sephacryl S-200 Column Chromatography
Gel filtration of antigen I/II was carried out on a Sephacryl S-200
column (88 cm.times.1.6 cm) and equilibrated with 0.1M-tris-HCl (pH
8.0), containing 4.0% SDS and 0.02% sodium azide. The column was
calibrated using .alpha.-chymotrypsinogen (25,700), soyabean
trypsin inhibitor (21,500), ribonuclease A (13,700), insulin
(6,000), insulin A chain (2,300) and glutamyl-glycyl-phenylalanine
(352) as standards. A 10 mg sample of the antigen I/II prepared as
under (b) above was dissolved in 1.5 ml of the elution buffer,
boiled for 10 minutes and cooled before it was loaded on to the
column. The effluent was monitored continuously at 254 nm and 1.5
ml fractions were collected. The absorbance of each fraction was
also measured at 230 nm at which wavelength they absorbed more
strongly than at any other wavelength in the u.v. spectrum.
Fractions recorded at 230 nm were pooled over the following
molecular weight ranges: (1) >33,000 (2) 33,000-21,500, (3)
21,500-13,700, (4) 13,700-8,000, (5) 8,000-3,000, (6) 3,000-2,300
and (7) <2,300. At absorbance of 230 nm, fraction 1,5 and 6
showed individual peaks, fraction 2 showed a shoulder on the major
peak of fraction 1 and fraction 3 was taken as the descending part
of the latter. Fraction 4 showed absorbance.
The pooled fractions were dialysed at room temperature in tubing
with a molecular weight cut-off of 1,000, in three steps as
follows: (i) against water for 12-24 hours with 2-3 changes; (ii)
against 40% methanol for 48 hours, with a change at 24 hours and
(iii) against water again for 48 hours, with several changes of
water. They were then lyophilised and reconstituted in 1.0 ml of
0.85% NaCl. The reconstituted materials were spun for 3 minutes at
25,000 g and the supernatants were collected. The protein content
and antigenicity were assayed for each of these 7 fractions as
indicated under section d(i) and the results are given in following
Table 2.
TABLE 2 ______________________________________ Protein Frac-
Content Single radial diffusion tion Molecular % with antisera to
SA No. Weight .mu.g Yield I/II I II III
______________________________________ 1 >33000 2031 20.31 + + +
- 2 <33000 > 21500 650 6.50 + + + + 3 <21500 > 13700
210 2.10 + + + Trace 4 <13700 > 8000 152 1.52 + + + - 5
<8000 > 3000 138 1.38 + + + - 6 <3000 > 2300 64 0.64 +
+ + - 7 <2300 60 0.60 + + + -
______________________________________
The fractions were then tested for efficiency of separation on both
15% polyacrylamide--6M urea and 7.5% polyacrylamide gels as
described under (c) above. Fraction 5 (mol. wt.<8000>3000)
showed a single band corresponding to a molecular weight of about
3800 as calculated from the gel. Fractions 6 and 7 also showed
bands equivalent to a molecular weight less than 4000 but the
protein concentrations were less than that in fraction 5 (see Table
2).
As indicated in Table 2, the SRID tests showed that antigen I and
II were present in all seven fractions and that antigen III was
absent in all but 2, with a trace in fraction 3. The presence of
antigens I and II but not III in fractions 5, 6 and 7 was confirmed
by the solid phase radio-immunoassay (see Clin. Exp. Immunol. 43
417-428). Of 6 Sephacryl column separations carried out, all
yielded the antigen of 3800 daltons in fraction 5 but this material
was detected in only 4/6 of the fractions 6 and 7.
It has also been found that if 0.1% SDS is used instead of 4% SDS
in the elution buffer, all but fractions 1 and 7 contain the 3800
dalton antigen I/II, with increasing intensity of staining from
fraction 2 to 6. However, with the 0.1% SDS the higher molecular
weight proteins were not separated completely. In contrast, with 4%
SDS the low molecular weight peptide was found only in fraction 5
and to a lesser extent in 6 and 7. This provides evidence, though
the invention is not to be bound by this theory, that the 3,800
dalton antigen is tightly bound to antigen I/II (185,000 daltons),
along with other proteins of intermediate molecular weights which
are demonstrable on the 15% polyacrylamide--6M urea gel.
(e) Analysis of Antigen X
(i) Amino acid analysis
The protein samples were hydrolysed at 110.degree. C. with 5.7M HCl
containing 0.1% phenol (w/v), in sealed evacuated tubes, after
flushing several times with N.sub.2 for 24 hours and analysed on a
Rank Hilger Chromaspek J 180 instrument. N-terminal residues were
determined by dansylation of the protein samples by the procedure
of Gray (Methods Enzymol 25 121-138 (1972)) and identification of
dansyl-amino acids by thin layer chromatography on polyamide layer
sheets (see Biochem. Biophys. Acta 133 369-370 (1967)). Dansyl
chloride and polyamide layer sheets were obtained from BDH
Chemicals, Poole, Dorset, UK.
The amino acid composition of the 3,800 dalton peptide is given in
following Table 3 and compared with that for the 185,000 dalton
antigen I/II.
TABLE 3 ______________________________________ 3,800 dalton peptide
185,000 dalton protein mol amino mol amino mol amino mol amino
acid/ acid/ acid/ acid/ mol peptide 1000 mol mol protein 1000 mol
______________________________________ Asp 3.1 78 172.6 99 Thr 3.0
76 165.4 95 Ser 3.5 87 136.5 79 Glu 2.2 55 182.9 105 Pro 1.4 35
106.6 61 Gly 7.3 182 119.5 69 Ala 6.1 152 204.9 118 Cys 0 0 0 0 Val
2.2 55 105.6 61 Met 0 0 0 0 Ile 2.0 51 82.3 47 Leu 3.4 85 105.3 61
Tyr 1.6 39 91.4 53 Phe 0.6 14 58.3 34 His 0.7 18 61.0 35 Lys 1.2 31
146.3 84 Arg 1.7 42 n.d. n.d.
______________________________________ n.d. not determined
From the above Table 3 it can be seen that the 3,800 dalton peptide
has a relatively lower content of charged amino acids but a
relatively higher content of non-polar residues than the 185,000
material. As no precautions were taken to convert cysteine and
methionine residues to stable derivatives prior to hyrolysis, no
significance can be ascribed to the absence of these two amino
acids in each preparation. Dansylation of the 3,800 mol. weight
fragment revealed two amino acids, alanine and glycine, as amino
terminal residues, together with trace amounts of a large number of
other amino acids. 38 or 40 amino acid residues were shown to make
up the antigen.
(ii) Proteolytic digestion of the antigen
42 .mu.g of the 3,800 d.antigen was taken up in 600 .mu.l of 0.1
M-tris/HCl and incubated at 37.degree. C. with pronase
(ennzyme:protein, 1:100 (w/w)). Samples of 100 .mu.l were taken at
0 min, 15 min, 30 min, 2 hours, 4 hours and 6 hours and boiled for
10 minutes. Samples were analysed for antigen I and II determinants
by the solid-phase radio-immunoassay technique (see Clin. Exp.
Immunol 43 417-428). Both the antigenic determinants I and II were
partially digested by 15 minutes' treatment and completely digested
by 30 minutes' treatment, as can be seen from Table 4 below which
shows the effect of pronase treatment expressed as a percentage
binding of .sub.125 I in the radioimmunoassay.
TABLE 4 ______________________________________ Duration of pronase
treatment (min) Antiserum to: 0 15 30 60
______________________________________ Antigenic determinant 1.46
0.63 0.22 0.23 I/II Antigenic determinant 1.40 0.75 0.28 0.38
Antigenic determinant 1.29 0.59 0.22 0.20 II Normal rabbit serum
0.05 0.02 0.05 0.03 ______________________________________
(iii) Carbohydrate analysis
The total monosaccharide contents of the 3,800 dalton material and
the 185,000 dalton material were determined by the method of Clamp
(see Biochem. Soc. Symp. 40 3-16 (1974)) on a Pye 204 gas
chromatograph. The column (1.5 m.times.4 mm) was packed with 10%
SE-30 (w.v.) on Chromosorb W HP AWDMCS, mesh size 100-120.
D(-)arabinose was used as standard. Two samples of the 3,800 dalton
material were found to contain 1.64% and 2.08% (w/w) of
monosaccharide in contrast to a mean (.+-.SD) of 6.57 (.+-.1.63)%
of 10 samples of the 185,000 dalton material.
(iv) Lipid analysis
Total lipid was extracted with chloroform and methanol (2:1, v/v)
by the method of Folch et al (see J. Biol Chem. 266 497-509 (1957))
and analysed by thin layer chromatography on plates coated with
silica gel H containing ammonium sulphate as described by Kaulen
(see Anal. Biochem 45 664-7 (1972)). Plates were developed with
hexane, diethyl ether and acetic acid (60:30:1, v/v) until the
solvent front reached the top of the plate. After thorough drying
the lipids were detected by exposure to iodine vapour for 3-5
minutes.
Thin layer chromatography revealed the presence of free fatty
acids, triglycerides and cholesterol esters, with R.sub.f values
slightly different from the mammalian lipid standards, in the
185,000 dalton antigen. As the lipid extract from 40 .mu.g of the
3,800 dalton material gave only a spot at the origin which was also
present in the control, antigen X was considered to be free of
lipids.
(f) Immunological properties of Antigen X
(i) In monkeys
A vaccine is prepared by dissolving the 3,800 dalton antigen
(Example ld(ii) fraction 5) in 0.85% w/v saline containing an equal
volume of Alhydrogel (Miles Laboratories Ltd) (aluminium hydroxide
suspension) to give a vaccine containing 10 .mu.g antigen per ml. 1
ml doses of this vaccine were injected subcutaneously in about 2 kg
rhesus monkeys. Within 14 days a very significant increase in the
IgG class of antibody was detected by radioimmunoassay, comparable
with that elicited by the 185,000 dalton antigen. This was
associated with a very marked increase in helper but not suppressor
function with the 3,800 daltong antigen, but both helper and
suppressor functions were elicited by the 185,000 daltons antigen,
as indicated by the results in Table 5 which follows. (The helper
and suppressor functions were generated by culture in Marbrook
flasks for 24 hours and then determining the number of Jerne
antibody forming cells, after cooperative culture for 4 days (see
Infect. Immun. 26 903, J. Immunol 124 2384 and Nature 292
770)).
TABLE 5 ______________________________________ Serum Antibodies
Helper Suppressor (% binding) Function (AFC)* Function (%) serum at
1:100 Day 3800 185,000 3800 185,000 3800 185,000
______________________________________ 0 5 0 12 0 0.6 0.6 7 242 208
11 94 0.4 1.9 14 177 ND 30 100 10.5 17.2 28 190 200 13 100 9.8 11.7
85 177 190 0 95 7.9 7.4 ______________________________________ *AFC
= antibody forming cells.
(ii) In man
The helper and suppressor functions of the 3,800 and 185,000
daltons preparations were compared using human lymphocytes in
vitro. This revealed (see results in Table 6 below) that whilst the
185,000 dalton antigen induced both help (with 1000 ng) and
suppression (with all other doses), the 3800 dalton antigen induced
help only with doses of 1-10,000 ng antigen as was observed in
rhesus monkeys.
TABLE 6
__________________________________________________________________________
Function 3800 dalton Strep. Antigen 185,000 dalton Strep. Antigen
Dosage 0 1 10 100 1000 10,000 0 1 10 100 1000 10,000
__________________________________________________________________________
% Help* 0 100 100 77 96 100 0 2 13 0 100 0 % Suppression* 2 12 2 12
9 28 12 88 100 96 2 100
__________________________________________________________________________
*% Help or Suppression calculated from the antibody forming cells
(AFC) given by mouse spleen helper cells: 277 .+-. 9 AFC and Strep.
antigen alone 53 .+-. 12 AFC.
(iii) Side reactions
Neither local nor systemic side reaction was observed. The blood
indices remained normal and there was no change in the haemoglobin
or in the red or white blood cell count. Antibodies to heart
homogenate were assayed by the solid phase radioimmunoassay and
were not detected.
EXAMPLE 2
1. Subcutaneous vaccination
A vaccine is prepared as described in Example 1, f(i). 1 ml doses
were injected subcutaneously in divided doses in the arm and thigh
of 4 young rhesus monkeys. Five control monkeys were injected with
saline only. All monkeys were then kept on a human type of diet,
containing about 15% sugar (Lehner, T., Challacombe S. J. and
Caldwell J. 1975, Archs, oral Biol. 20, 299). Two of the monkeys
were reinjected 2 weeks later with 10 .mu.g of a similar vaccine
but without the aluminium hydroxide adjuvant. The monkeys were
examined regularly for dental caries, serum antibodies and for
colonisation with Strep. mutans.
The results of this examination is given in Table 7 and show
significant reduction in carious lesions in the immunised monkeys,
mean .+-. standard error (0.25.+-.0.25) as compared with the
control animals (2.4.+-.0.75) after a period of about 11/2 years.
Analysis by the Student's test showed these results to be
significant at the 5% level (t=2.453, d.f.7, p<0.05).
There was a significant increase in the serum IgG antibody titre
assayed by a radioimmunoassay (cpm) in the immunised (2788.+-.884),
as compared with the control monkeys (472.+-.123). However, the
difference in the number of colony forming units failed to reach a
significant level.
2. Topical gingival vaccination
A topical vaccine was prepared by dissolving 3800 dalton antigen X
in 0.85% w/v saline at a concentration of 500 .mu.g per ml of
saline solution and to increase its permeability through the
gingival crevicular epithelium 50% dimethyl sulphoxide (DMSO) was
added to the solution. About 200 .mu.g of this vaccine was applied
topically to the gingival sulci of the upper and lower jaws of 2
young rhesus monkeys and kept in place for about 5 minutes by means
of a preformed silicone rubber appliance which fitted exactly the
teeth and gums. The appliance was made by taking an impression of
the upper and lower jaw with Optosil (Bayer Dental), constructing a
tray with cold cure acrylic, taking further impressions with the
same material and then lining it with Xantopren plus material
(Bayer Dental). On inserting the appliance, fine digital pressure
was applied 30 times in rapid succession, in order to facilitate
the vaccine to reach the crevicular epithelium. This procedure was
repeated 4 times over a period of a month, repeated twice at 3
months and once at 5 months.
Two control monkeys were injected subcutaneously with saline. All
monkeys were placed on the human type of diet. The monkeys were
examined for dental caries, serum and gingival fluid, IgG
antibodies, salivary IgA antibodies and colonisation of Strep.
mutans.
The results of 2 immunised and 2 control monkeys (see Table 8)
suggest that gingival immunisation prevents dental caries by the
presence of gingival fluid IgG class of antibodies and salivary IgA
class of antibodies. These are associated with a marked decrease in
the number of colonies of Strep. mutans. An important feature is
the absence of a significant increase in serum antibodies to Strep.
mutans. Hence, gingival immunisation with antigen X induces
protection against dental caries, in the absence of serum
antibodies and therefore any systemic side effects. There were no
detectable local changes in the gingiva.
TABLE 7 ______________________________________ Serum IgG Dose of SA
No. of carious Antibody titre Rhesus Monkey injected lesions (cpm)
______________________________________ 1 10 .mu.g .times. 2 1 5204
2 10 .mu.g .times. 2 0 2979 3 10 .mu.g .times. 1 0 1725 4 10 .mu.g
.times. 1 0 1246 *0.25 (.+-.0.25) *2788 (.+-.884) 5 0 2 290 6 0 4
131 7 0 0 759 8 0 4 440 9 0 2 740 *2.4 (.+-.0.75) *472 (.+-.123)
______________________________________ *Mean (.+-.standard
error)
TABLE 8
__________________________________________________________________________
Colony forming No. of gingival No. of carious units of Gingival
fluid Rhesus monkey immunissations lesions Strep. mutans Serum IgG*
IgG* Salivary IgA*
__________________________________________________________________________
1 .times.7 0 10.5 0 42 44.2 2 .times.7 0 15.8 0 50.6 36.4 3 0 7
59.8 0 0 0 4 0 2 72.5 14.0 0 0
__________________________________________________________________________
*Increase in antibody titre above the preimmunised level assayed by
radioimmunoassay (Smith R. and Lehner T., Clin. exp. Immunol. 43
417-428) and given in cpm.
EXAMPLE 3
Preparation of antigen X by enzyme digestion
This Example describes the production of antigen X using a
bacterial protease. The starting material for these protease
digestions was the 185,000 dalton molecular weight antigen which
was eluted in the void volume of the Sephacryl 200 gel filtration
column run in 4% SDS, as described in Example 1 d(ii). SDS gradient
polyacrylamide gel electrophoresis (PAGE) of this starting material
failed to detect any bands with a molecular weight lower than
approximately 3500. The protease used was Staphylococcus Aureus V8
protease (Miles Laboratories Ltd.) at a weight:weight ratio of 1:20
of enzyme:185,000 daltons antigen I/II.
The starting antigen was dissolved in 50 mM NH.sub.4 HCO.sub.3, pH
7.8, at a concentration of 2 mg/ml and boiled for 4 minutes. After
cooling the solution to 37.degree. C., the appropriate amount of
protease, dissolved in the same buffer was added. The mixture was
shaken and kept at 37.degree. C. for the duration of the digestion.
Digestion was continued for up to 17 hours at which time the enzyme
was inactivated by boiling the solution. The resulting digested
antigen was assayed by the solid phase radioimmunoassay for antigen
activity and the molecular weight of the digestion products were
investigated using SDS gradient PAGE and immuno-blotting. The major
bands were at 4, 8, 10 and 12 k daltons and the 4 k daltons
material was then separated by elution from SDS-polyacrylamide
gels. One mg of protease-digested antigen was loaded onto an
SDS-gradient polyacrylamide gel and electrophoresed. The gel was
then sliced according to the prestained molecular weight markers
(Bethesda Research Laboratories Inc. USA), and each slice was
extracted for 24 hours at 37.degree. C. with 0.01M Tris-HCl buffer
(pH 8), containing 0.05% w/v SDS and 1 mM sodium azide. The
extracts were dialysed extensively against methanol and water,
lyophilised and reconstituted in 0.85% w/v NaCl. In this way a
fraction of molecular weight 3800-4500 was isolated.
* * * * *